Hematopoietic stem cell sources include frozen bone marrow, mobilized hematopoietic progenitor cells, and umbilical cord blood. These stem cells can be frozen viably for subsequent thawing and transplantation for both autologous and allogeneic hematopoietic cell transplantation. About 50% of patients with diseases curable only by hematopoietic stem cell transplantation will not have a HLA-matched sibling to serve as a stem cell donor.
Matched unrelated donor transplants and transplants using umbilical cord blood (UCB) have been increasingly used as a source of hematopoietic stem cells to treat patients with hematological disorders requiring an allogeneic stem cell transplant (SCT) who lack an HLA matched sibling or unrelated donor. The advantages to UCB transplantation include the ease and rapidity of availability of the UCB unit (more than 100,000 frozen UCB units are available in public registries), the ability to effectively use a less-than-perfect HLA match, and lower rates of graft-versus-host disease (GVHD) compared to mismatched bone marrow or peripheral blood stem cell transplants. GVHD, an orphan disease, is a major obstacle to successful allogeneic stem cell transplantation. With UCB transplants, even a 4/6 or 5/6 HLA-matched donor can be used safely. Rates of GVHD with 4/6 or 5/6 matched UCB have been comparable to that of matched bone marrow transplantation. Therefore, UCB transplants appear to be an effective option for patients with disease curable by allogeneic stem cell transplant (SCT) who lack a suitable HLA-matched donor.
However, despite the reduced GVHD associated with HLA mismatching, severe GVHD resulting in morbidity and death still occurs in up to 15% of recipients of a UCB transplant. Furthermore, because the majority of T-cells contained within an UCB unit are naive to viral pathogens, severe morbidity and death associated with viral reactivation of adenovirus, cytomegalovirus, and Epstein Barr virus (EBV) occurs more commonly after UCB transplantation compared to peripheral blood or bone marrow transplants. There is a need to develop a solution to suppress the alloreactivity of infused donor T-lymphocytes to reduce the substantial GVHD-associated morbidity and mortality that occurs following UCB transplantation while simultaneously providing immune competent viral specific T-cells to prevent viral reactivation.
The use of adoptive natural killer (NK) cell infusions following UCB transplantation offers the possibility of reducing GVHD while enhancing a potent graft-versus-tumor (GVT) effect. Data suggest that the NK cells protect recipients from GVHD in the setting of killer IgG-like receptor (KIR) ligand incompatibility. In humans, this protective effect is most evident with MHC mismatched transplantation, usually following in vivo or in vitro T-cell depletion. In MHC mismatched murine transplant models, lethal GVHD is reduced following the adoptive infusion of KIR ligand mismatched NK cells. Adoptively infused NK cells, while reducing GVHD, simultaneously mediate an anti-tumor effect against tumor cells. These data support the potential for an adoptive infusion of alloreactive NK-cells to reduce the incidence of GVHD and tumor relapse in humans undergoing T-cell-replete allogeneic SCT. Furthermore, they suggest that an adoptive infusion of in vitro expanded NK cells isolated from the same UCB unit used for SCT might likewise reduce GVHD and improve survival following UCB transplantation. NK cells isolated from thawed umbilical cord blood units can be expanded in vitro by 100 to 1000 fold, numbers that would be sufficient to be used for adoptive transfer into recipients of an umbilical cord blood transplant.
Viral reactive T-cells capable of killing adenovirus, cytomegalovirus, and Epstein Barr virus (EBV) can be expanded in vitro from peripheral blood mononuclear cells (PBMC). Using dendritic cells generated from human CD34+ cells or monocytes transduced with a adenoviral vectors encoding CMV antigens, CD4+ T-cells and cytotoxic T-cells that are adenoviral and CMV reactive have been stimulated and expanded. By further stimulating these T-cells with EBV transformed B-cells, EBV-Reactive T-cells can likewise be expanded in vitro and can be used to prevent and treat EBV lympho-proliferative disorder. CD4+ T-cells and cytotoxic T-cells that are adenoviral, CMV-reactive, and EBV-reactive can be generated in vitro from mononuclkaer cells obtained from UCB units. However, this process of viral reactive T-cell expansion typically takes 6-8 weeks. Unfortunately, many life-threatening viral reactivations occur within the first 6 weeks of UCB transplantation, so expansion of viral reactive T-cells from mononuclear cells taken from a thawed UCB unit (at the time of transplantation) would not be available in time to prevent a majority of viral reactivations associated with UCB transplantation.
Furthermore, data suggest that adoptive NK cell infusions must be given early, at the same time the hematopoietic stem cells are transplanted, in order to kill host antigen presenting cells and thus prevent GVHD. With standard PBSC or marrow transplants, the donor is typically available 2-4 weeks before transplantation to donate lymphocytes to expand NK cells or to collect T-cells should a subsequent donor lymphocyte infusion be required to treat disease relapse. On the other hand, with frozen UCB units, adoptive NK cell and/or T-cell infusions at the time of transplantation are currently not possible, because the entire UCB unit is defrosted at one time and transplanted, eliminating the donor cell source from which these cells could be expanded. Although a portion of the thawed UCB unit could be set aside at the time of thawing and preserved to expand NK cells or T-cells, in vitro NK and T-cell expansions require 3-8 weeks to expand a sufficient number of cells to prevent GVHD.
There have been previous approaches to this problem by providing storage containers with separate storage compartments to allow different portions of a frozen cell sample to be thawed at different times. See, e.g., U.S. Patents and applications U.S. Pat. No. 6,491,678, 2005/0084838, and 2004/0097862, and PCT Applications 2007/059084 and 97/49959. These methods require separation of the cell sample portions before freezing. Most frozen UCB units are contained in a bag with either a single compartment or two compartments, the two compartments respectively containing, for example, 5 ml and 20 ml of frozen umbilical cord blood. For these types of UCB units, the aforementioned solutions do not permit thawing or selective access to only a small portion of the UCB unit, i.e., 1-2 ml.
Currently, no method exists that is capable of selectively accessing and/or partially thawing one portion of a frozen UCB unit without compromising the integrity, sterility, or viability of the unthawed portion, e.g., 2-4 weeks prior to a transplant, in order to expand NK cells, T-cells, or hematopoietic stem cells in vitro, for adoptive infusion at the time of transplantation of the remaining portion of the UCB unit. Such a method could allow these expanded cells (i.e. hematopoietic stem cells, NK cells, T-cells, etc.) to be used to potentiate graft-versus leukemia effects, prevent GVHD, and to prevent or treat viral infections in the post-UCB transplant period. Such a method could permit adoptive cellular immunotherapy using immune cells from the same UCB unit that is transplanted to more rapidly restore lympho-hematopoietic function. The ability to selectively thaw a portion of a UCB unit has utility in numerous other therapeutic modalities.